1. Field of the Invention
The present invention relates to DNA constructs and plants incorporating them. In particular, it relates to expression cassettes and promoter sequences for the expression of genes in plants.
2. Description of Related Art
Gene expression is controlled by regions upstream (5xe2x80x2) of the protein encoding region, commonly referred to as the xe2x80x9cpromoterxe2x80x9d. A promoter may be constitutive, tissue-specific, developmentally-programmed or inducible.
Manipulation of crop plants to improve characteristics (such as productivity or quality) requires the expression of foreign or endogenous genes in plant tissues. Such genetic manipulation therefore relies on the availability of means to control gene expression as required; for example, on the availability and use of suitable promoters which are effective in plants. It is advantageous to have the choice of a variety of different promoters so that the most suitable promoter may be selected for a particular gene, construct, cell, tissue, plant or environment. A range of promoters are known to be operative in plants.
Within the promoter region there are several domains which are necessary for full function of the promoter. The first of these domains lies immediately upstream of the structural gene and forms the xe2x80x9ccore promoter regionxe2x80x9d containing consensus sequences, normally 70 base pairs immediately upstream of the gene. The core promoter region contains the characteristic CAAT and TATA boxes plus surrounding sequences, and represents a transcription initiation sequence which defines the transcription start point for the structural gene. The precise length of the core promoter region is indefinite but it is usually well-recognisable. Such a region is normally present, with some variation, in all promoters. The base sequences lying between the various well-characterised xe2x80x9cboxesxe2x80x9d appear not to be of great importance.
The presence of the core promoter region defines a sequence as being a promoter: if the region is absent, the promoter is non-functional. Furthermore, the core promoter region is insufficient to provide full promoter activity. A series of regulatory sequences upstream of the core constitute the remainder of the promoter. The regulatory sequences determine expression level, the spatial and temporal pattern of expression and, for an important subset of promoters, expression under inductive conditions.
Several naturally-occurring promoters and associated gene expression systems are known. The best characterised regulatory systems are those of bacteria in which the specific interactions between DNA-binding proteins (repressors) and the target DNA sequences (operators) results in the negative repression of gene activity.
The alcA/alcR gene activation system from the fungus Aspergillus nidulans is also well characterised. The ethanol utilization pathway in A nidulans is responsible for the degradation of alcohols and aldehydes. Three genes have been shown to be involved in the ethanol utilization pathway. Genes alcA and alcR have been shown to lie close together on linkage group VII and aldA maps to linkage group VIII (Pateman J H et al, 1984, Proc. Soc. Lond, B217:243-264; Sealy-Lewis H M and Lockington R A, 1984, Curr. Genet, 8:253-259). Gene alcA encodes ADHI in A nidulans and aldA encodes AldDH, the second enzyme responsible for ethanol utilization. The expression of both alcA and aldA are induced by ethanol and a number of other inducers (Creaser E H et al, 1984, Biochemical J, 255:449-454) via the transcription activator alcR. The alcR gene and a co-inducer are responsible for the expression of alcA and aldA since a number of mutations and deletions in alcR result in the pleiotropic loss of ADHI and aldDH (Felenbok B et al, 1988, Gene, 73:385-396; Pateman et al, 1984; Sealy-Lewis and Lockington, 1984). The ALCR protein activates expression from alcA by binding to three specific sites in the alcA promoter (Kulmberg P et al, 1992, J. Biol. Chem, 267:21146-21153).
The alcR gene was cloned (Lockington R A et al, 1985, Gene, 33:137-149) and sequenced (Felenbok et al, 1988). The expression of the alcR gene is inducible, autoregulated and subject to glucose repression mediated by the CREA repressor (Bailey C and Arst H N, 1975, Eur. J. Biochem, 51:573-577; Lockington R A et al, 1987, Mol. Microbiology, 1:275-281; Dowzer C E A and Kelly J M, 1989, Curr. Genet, 15:457-459; Dowzer C E A and Kelly J M, 1991, Mol. Cell. Biol, 11:5701-5709). The ALCR regulatory protein contains 6 cysteines near its N terminus coordinated in a zinc binuclear cluster (Kulmberg P et al, 1991, FEBS Letts, 280:11-16). This cluster is related to highly conserved DNA binding domains found in transcription factors of other ascomycetes. Transcription factors GAL4 and LAC9 have been shown to have binuclear complexes which have a cloverleaf type structure containing two Zn(II) atoms (Pan T and Coleman J E, 1990, Biochemistry, 29:3023-3029; Halvorsen Y D C et al, 1990, J. Biol. Chem, 265:13283-13289). The structure of ALCR is similar to this type except for the presence of an asymmetrical loop of 16 residues between Cys-3 and Cys-4. ALCR positively activates expression of itself by binding to two specific sites in its promoter region (Kulmberg P et al, 1992, Molec. Cell. Biol, 12:1932-1939).
The regulation of the three genes, alcR, alcA and aldA, involved in the ethanol utilization pathway is at the level of transcription (Lockington et al, 1987; Gwynne D et al, 1987, Gene, 51:205-216; Pickett et al, 1987, Gene, 51:217-226).
There are two other alcohol dehydrogenases present in A nidulans. ADHII is present in mycelia grown in non-induced media and is repressible by the presence of ethanol. ADHII is encoded by alcB and is also under the control of alcR (Sealy-Lewis and Lockington, 1984). A third alcohol dehydrogenase has also been cloned by complementation with a adh-strain of S cerevisiae. This gene alcC, maps to linkage group VII but is unlinked to alcA and alcR. The gene, alcC, encodes ADHIII and utilizes ethanol extremely weakly (McKnight G L et al, 1985, EMBO J, 4:2094-2099). ADHIII has been shown to be involved in the survival of A nidulans during periods of anaerobic stress. The expression of alcC is not repressed by the presence of glucose, suggesting that it may not be under the control of alcR (Roland L J and Stromer J N, 1986, Mol. Cell. Biol, 6:3368-3372).
In summary, A nidulans expresses the enzyme alcohol dehydrogenase I (ADH1) encoded by the gene alcA only when it is grown in the presence of various alcohols and ketones. The induction is relayed through a regulator protein encoded by the alcR gene and constitutively expressed. In the presence of inducer (alcohol or ketone), the regulator protein activates the expression of the alcA gene. The regulator protein also stimulates expression of itself in the presence of inducer. This means that high levels of the ADH1enzyme are produced under inducing conditions (ie when alcohol or ketone are present). Conversely, the alcA gene and its product, ADH1, are not expressed in the absence of inducer. Expression of alcA and production of the enzyme is also repressed in the presence of glucose.
Thus the alcA gene promoter is an inducible promoter, activated by the alcR regulator protein in the presence of inducer (ie by the protein/alcohol or protein/ketone combination). The alcR and alcA genes (including the respective promoters) have been cloned and sequenced (Lockington R A et al, 1985, Gene, 33:137-149; Felenbok B et al, 1988, Gene, 73:385-396; Gwynne et al, 1987, Gene, 51:205-216).
Alcohol dehydrogenase (adh) genes have been investigated in certain plant species. In maize and other cereals they are switched on by anaerobic conditions. The promoter region of adh genes from maize contains a 300 bp regulatory element necessary for expression under anaerobic conditions. However, no equivalent to the alcR regulator protein has been found in any plant. Hence the alcR/alcA type of gene regulator system is not known in plants. Constitutive expression of alcR in plant cells does not result in the activation of endogenous adh activity.
The knowledge of mechanisms by which gene expression is regulated in eukaryotes is much less detailed than the knowledge of bacterial systems. In yeast and mammalian cells a large number of binding sites for putative regulatory proteins have been identified in promoter sequences, and in some cases the proteins responsible have also been isolated. However, only in a few instances are the molecular details known of the protein-DNA interactions and the mechanism by which transcription is regulated. In plants, regulation of gene expression is understood at only a rudimentary level. Several regulatory elements have been identified in promoter sequences, and some regulatory proteins examined at a preliminary level. Many such proteins have yet to be isolated and the details of the mechanisms involved have yet to be elucidated.
The control of expression of heterologous genes in plant tissues is important for successful genetic manipulation of plants to alter and/or improve phenotypic characteristics. Promoters and/or regulatory components from bacteria, viruses, fungi and plants have been used to control gene expression in plant cells. For example, two well-characterised bacterial operator-repressor systems have been used to show negative (down) regulation of gene expression in plant cells. A modified bacterial tet operator-repressor system has been shown to repress gene expression in electroporated plant cells. Gene expression from the CAT gene was reduced by the placing of tet operators in the 35S promoter (Gatz and Quail, 1988, Proc. Natl. Acad. Sci. USA, 85:1394-1397). Functional tet repressor expressed from an integrated gene also represses expression from an integrated xcex2-glucuronidase gene (gus) driven by the 35S promoter containing two inserted tet operators (Gatz et al, 1991, Mol. Gen. Genet, 227:229-237).
The lac operator-repressor system has been used to repress gene expression in tobacco cells. Functional LacI protein expressed from a stably integrated lacI gene repressed expression from a stably integrated gus gene controlled by the maize chlorophyll a/b binding protein (CAB) promoter containing a lac operator. Derepression of gus activity occurred when the cells were incubated with the inducer iso-propyl thiogalactoside (IPTG) showing that repression of gene activity is specifically due to the lacI gene product (Wilde R J et al, 1992, EMBO J, 11:1251-1259).
As stated above, successful genetic manipulation relies on the availability of means to control gene expression as required. The scientist may use a suitable expression cassette (incorporating one or more promoters and other components) to regulate gene expression in the desired manner. The ability to enhance or reduce gene expression to achieve a desired phenotypic effect according to external circumstances is particularly advantageous. We now provide a novel plant gene expression cassette which allows external activation of target gene expression within plants.
According to a first aspect of the invention, there is provided a chemically-inducible plant gene expression cassette comprising a first promoter operatively linked to a regulator sequence which encodes a regulator protein, and an inducible promoter operatively linked to a target gene, the inducible promoter being activated by the regulator protein in the presence of an effective exogenous inducer whereby application of the inducer causes expression of the target gene.
The first promoter may be constitutive, or tissue-specific, developmentally-programmed or even inducible. The regulator-sequence is preferably the alcR gene obtainable from Aspergillus nidulans, encoding the alcR regulator protein.
The inducible promoter is preferably the alcA gene promoter obtainable from Aspergillus nidulans, or a xe2x80x9cchimericxe2x80x9d promoter derived from the regulatory sequences of the alcA promoter and the core promoter region from a gene promoter which operates in plant cells (including any plant gene promoter). The alcA promoter or a related xe2x80x9cchimericxe2x80x9d promoter is activated by the alcR regulator protein when an alcohol or ketone inducer is applied.
The inducible promoter may also be derived from the aldA gene promoter, the alcB gene promoter or the alcC gene promoter obtainable from Aspergillus nidulans. 
The target gene may be an endogenous plant gene or a foreign gene, and may be a single gene or a series of genes. The target gene sequence encodes at least part of a functional protein or an antisense sequence.
The inducer may be any effective chemical (such as an alcohol or ketone). Suitable chemicals for use with an alcA/alcR-derived cassette include those listed by Creaser et al (1984, Biochem J, 225, 449-454) such as butan-2-one (ethyl methyl ketone), cylcohexanone, acetone, butan-2-ol, 3-oxobutyric acid, propan-2-ol, ethanol.
The gene expression cassette is responsive to an applied exogenous chemical inducer enabling external activation of expression of the target gene regulated by the cassette. The expression cassette is highly regulated and suitable for general use in plants.
The two parts of the expression cassette may be on the same construct or on separate constructs. The first part comprises the regulator cDNA or gene sequence subcloned into an expression vector with a plant-operative promoter driving its expression. The second part comprises at least part of an inducible promoter which controls expression of a downstream target gene. In the presence of a suitable inducer, the regulator protein produced by the first part of the cassette will activate the expression of the target gene by stimulating the inducible promoter in the second part of the cassette.
As an example of the invention, a gene expression cassette was constructed comprising the Aspergillus nidulans alcR gene under control of a constitutive promoter and an alcA:35S inducible xe2x80x9cchimericxe2x80x9d promoter linked to the chloramphenicol acetyl transferase (CAT) gene from E coli. Constructs representing the two parts of this expression cassette were tested in Aspergillus nidulans. For example:
(1) An alcR cDNA was expressed in A nidulans by placing it downstream from the CaMV 35S or nos promoter. Transformants of an alcRxe2x88x92strain were shown to be wild type (alcR+) with respect to growth on ethanol as sole carbon source, showing that the alcR construct was expressing the regulator protein.
(2) A construct containing a CAT gene downstream from the alcA:35S xe2x80x9cchimericxe2x80x9d promoter was tested in a (alcR+) A nidulans strain. The CAT gene was used as the target gene because it is a suitable reporter gene for monitoring gene expression. CAT activity was alcohol-inducible (ie repressor protein plus inducer were activating the xe2x80x9cchimericxe2x80x9d promoter), showing that the xe2x80x9cchimericxe2x80x9d promoter is functional.
(3) The regulator alcR construct and the alcA reporter construct were co-transformed into an alcR xe2x88x92Axe2x88x92deletion strain. Any expression of the CAT reporter gene is induced by the alcR regulator protein produced by the regulator construct, without interference from a wild-type regulator protein. The alcA:35S chimeric promoter is inducible: the promoter drives expression of the CAT reporter gene when the regulator protein and inducer are present. Thus the gene expression cassette (comprising the alcR and alcA constructs) is functional and chemically-inducible (ie subject to external activation).
This invention is based on the construction of the above type of expression cassette and on our recognition that the functional elements of the cassette are suitable for application in other organisms, particularly in plants (such as tobacco, tomato, canola, sugarbeet, sunflower, maize, or wheat).
In practice the construct or constructs comprising the expression cassette of the invention will be inserted into a plant by transformation. Expression of target genes in the construct, being under control of the chemically switchable promoter of the invention, may then be activated by the application of a chemical inducer to the plant.
Any transformation method suitable for the target plant or plant cells may be employed, including infection by Agrobacterium tumefaciens containing recombinant Ti plasmids, electroporation, microinjection of cells and protoplasts, microprojectile transformation and pollen tube transformation. The transformed cells may then in suitable cases be regenerated into whole plants in which the new nuclear material is stably incorporated into the genome. Both transformed monocot and dicot plants may be obtained in this way.
Examples of genetically modified plants which may be produced include field crops, cereals, fruit and vegetables such as: canola, sunflower, tobacco, sugarbeet, cotton, soya, maize, wheat, barley, rice, sorghum, tomatoes, mangoes, peaches, apples, pears, strawberries, bananas, melons, potatoes, carrot, lettuce, cabbage, onion.
The invention further provides a plant cell containing a gene expression cassette according to the invention. The gene expression cassette may be stably incorporated in the plant""s genome by transformation. The invention also provides a plant tissue or a plant comprising such cells, and plants or seeds derived therefrom.
The invention further provides a method for controlling plant gene expression comprising transforming a plant cell with a chemically-inducible plant gene expression cassette which has a first promoter operatively linked to a regulator sequence which encodes a regulator protein, and an inducible promoter operatively linked to a target gene, the inducible promoter being activated by the regulator protein in the presence of an effective exogenous inducer whereby application of the inducer causes expression of the target gene.
As an example of the invention, we have demonstrated the functioning of alcR/alcA constructs in plant cells, and have shown that the system is useful as an inducible plant gene expression cassette. Regulator and reporter constructs were transformed separately and together into maize plant protoplasts for transient gene expression assays. Expression of the CAT gene from the alcA promoter in maize protoplasts incubated in the presence of ethanol (inducer) is dependent on the presence of the alcR gene (expressing the regulator protein). Stably transformed tobacco plants containing the alcR/alcA-CAT expression cassette were also produced. High levels of reporter (CAT) gene expression were only obtained in the presence of the alcR regulator gene product and following chemical induction by cyclohexanone. High levels of activity from the alcA-CAT reporter gene construct were not obtained under a range of growth conditions such as anaerobis, indicating that reporter gene expression was not induced by conditions that might normally be expected to induce plant alcohol dehydrogenases.
The inducible promoter forming part of the gene expression cassette according to the invention may be a xe2x80x9cchimericxe2x80x9d promoter sequence, created as described above by fusing heterologous upstream and downstream regions. Such a chimeric promoter may have independent utility as an alternative plant promoter for use in genetic manipulation. Comai et al have previously described a chimeric plant promoter combining elements of the CaMV35S and the mannopine synthase (mas) promoters (1990, Plant Mol Biol, 15:373-381). In electroporated tobacco cells, the yeast GAL4 protein has been shown to stimulate CAT gene expression controlled from a 35S promoter which had GAL4 binding sites upstream from the TATA box (Ma J et al, 1988, Nature, 334:631-633).
According to a second aspect of the invention, there is provided a chimeric promoter comprising an upstream region containing a promoter regulatory sequence and a downstream region containing a transcription initiation sequence, characterised in that said upstream and downstream regions are heterologous.
The upstream region may contain a constitutive, a tissue-specific, a developmentally-programmed or an inducible promoter regulatory sequence. There may be one or more regulatory sequences in the upstream region. The downstream region is a core promoter region. The upstream and downstream regions correspond to sequences isolated respectively from different sources. The upstream or downstream regions are fused together. The upstream and/or downstream regions may be synthesised.
The upstream sequence is preferably derived from the inducible alcA gene promoter obtainable from Aspergillus nidulans. The upstream sequence may also be derived from the aldA, alcB or alcC gene promoters obtainable from Aspergillus nidulans. The downstream sequence may be derived from the core promoter region of any plant-operative promoter (such as the CaMV35S promoter, MFS14, MFS18, GSTII-27, pMR7, Polygalacturonase promoters), or may be synthesised from consensus sequences.
The chimeric promoter may be operatively linked to one or more target gene sequences encoding at least part of a functional protein or an antisense RNA. The target gene may be any endogenous plant gene or any foreign gene.
In practice the promoter of the invention will be inserted as a promoter sequence in a recombinant gene construct destined for use in a plant. The construct will then be inserted into the plant by transformation. Any plant species may be transformed with the construct, and any suitable transformation method may be employed.
The invention further provides a plant cell containing a chimeric promoter according to the invention. The chimeric promoter may be stably incorporated in the plant""s genome by transformation. The invention also provides a plant tissue or a plant comprising such cells, and plants or seeds derived therefrom.
As an example of the invention, a chimeric gene promoter sequence has been produced by fusing a regulatory region from the Aspergillus nidulans alcA gene promoter to part of the xe2x88x9270 core region of the Cauliflower mosaic virus (CaMV) 35S promoter (see Example 2 and FIG. 3). The alcA:35S chimeric promoter sequence was linked to the chloramphenicol acetyl transferase (CAT) gene from E coli and transformed into an (alcR+) A nidulans strain. The CAT gene was used as a reporter gene to monitor gene expression. CAT production indicated that the chimeric promoter was functioning correctly (see Example 3).
The alcA:35S chimeric promoter is also suitable for use in any plant. Transient expression assays in maize protoplasts (Example 4) and expression from stably-incorporated genes in tobacco (Example 5) have shown that the alcA:35S promoter is fully functional in plant cells.